Xiaojian Hu

A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants of sfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant of sfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. The sfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.

Associations of protein 4.2 with band 3 and ankyrin.

Protein-protein and protein-lipid interactions are thought to play the vital role in maintenance and deformation of red blood cell (RBC) membrane. Protein 4.2, a 76-KDa peripheral protein, binds to the cytoplasmic domain of band 3 (CDB3) and also interacts with ankyrin in RBCs. In order to explore the characteristics of protein 4.2–CDB3–ankyrin interactions, three protein 4.2-derived recombinant proteins encompassing amino acid residues 31–200, 1–300, and 187–260 respectively were expressed in Escherichia coli. Their interactions with CDB3 and ankyrin were investigated by using Far-Western blot and pull-down assay. The results showed that the CDB3-binding site of protein 4.2 is located in the region of residues 200–211 and the ankyrin-binding site is located in the region of residues 187–200 of protein 4.2. Our findings also suggested that the ankyrin D34 domain can interact directly with protein 4.2. The proper tertiary structures of these protein 4.2 fragments are essential for protein 4.2–ankyrin interaction. Meanwhile, ankyrin can enhance the interaction between protein 4.2 and CDB3.

Arginase‐flotillin interaction brings arginase to red blood cell membrane

Flotillin‐1 and arginase are both up‐regulated in red blood cell membrane of type 2 diabetic patients. For studying why the soluble arginase can bind to the membrane and whether such binding would modify arginase activity, the arginase1 and related proteins were cloned and expressed. The results showed that flotillin‐1 can interact with arginase1, and hence arginase activity was up‐regulated by 26.8%. It was estimated that about 61% of arginase1 is bound to the membrane mediated by flotillin‐1. The arginase activity in diabetic patients was significantly higher than that of the controls (752.4 ± 38.5 U/mg protein vs 486.7 ± 28.7 U/mg protein).

A novel method for high-level production of psychrophilic TAB5 alkaline phosphatase

Heat labile alkaline phosphatases (APs) are widely used in biomedical research for they can easily be heat inactivated once they have done their job. Here we reported a novel method for high-level production of recombinant psychrophilic Antarctic bacterium strain TAB5 alkaline phosphatase (TAP) in Escherichia coli. We synthesized the whole TAP gene according to the synonymous codon choice that is optimal for the E. coli translational system. Then the gene was cloned into pT7 expression vector, expressed in BL21 (DE3) pLysS strain by auto-induction system. The recombinant protein was purified by Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 90.9 mg protein from 16.2 g wet cells in 1L culture medium with the purity over 99%, 340 times as many mg/L (and 21 times the mg/g cells) compared to previous methods. The dephosphorylation activity assay showed that the purified recombinant TAP has similar activity to calf intestinal alkaline phosphatase in room temperature, and it can be totally inactivated by treatment at 60°C for 15 min. Our research provides a novel method for high-level expression, purification and characterization of TAP which is sufficient for high throughput genome analysis and may replace the widely used shrimp AP because of its low cost.

Crystallization and Preliminary X-Ray Analysis of Sau3AI C-Terminal 232-419 Amino Acids Fragment

The C-terminal 232-419 amino acids fragment of endonuclease Sau3AI has been successfully expressed in Escherichia coli with 6 His at its N-terminal. After purification and crystallization, one completed 2.8 A data set was collected using a Rigaku R-AXIS IV++ diffractometer. The plate-like crystals belong to orthorhombic space group P2(1)2(1)2(1) with the cell dimension of a = 34.75, b = 76.82, c = 123.59A and contain one molecule per asymmetric unit.

Rational design of a pH‐insensitive cyan fluorescent protein CyPet2 based on the CyPet crystal structure

The emission spectrum of widely used CyPet is pH‐sensitive. In order to synthesize a pH‐insensitive cyan fluorescent protein by rational design, we solved the crystal structures of CyPet under different pH conditions. The indole group of the CyPet chromophore adopts a cis‐coplanar conformation in acidic and neutral conditions, while it converts to trans‐coplanar under basic conditions. His148 and Glu222 play a vital role in this isomerization. The pH‐sensitive chromophore isomerization and change in the emission spectrum can be explained by the coexistence of several different fluorescent states. We trap the chromophore in the trans conformation by A167I mutation (CyPet2), which also prevents the multiconformation of the seventh β‐strand. CyPet2 exhibits an unchanged emission spectral shape as a function of pH.

Expression, Purification, Crystallization and Preliminary X-Ray Analysis of Cyan Fluorescent Protein CyPet

The technique of fluorescence (or Förster) resonance energy transfer (FRET) is widely used to observe bimolecular interaction in living cells. Cyan and yellow fluorescent proteins are the most widely used pair in FRET analysis. CyPet and YPet are two newly optimized fluorescent proteins that have much better dynamic range and sensitivity than CFP/YFP pair, although the crystallographic structure and the mechanism of better fluorescent characteristics of CyPet are still unknown. We have expressed the cyan fluorescent protein CyPet using pT7 prokaryocyte expression system in Escherichia coli strain Rosetta (DE3) pLysS by auto-induction. After purification, the recombinant CyPet protein was crystallized by hanging drop vapor diffusion technique and could diffract to 2.55A resolution. The data showed that the orthorhombic CyPet crystal was in space group P212121 with unit cell parameters (51.55, 61.53, 63.36) and contained one molecule in one asymmetric unit.

Crystallization and Preliminary X-Ray Analysis of Fluorescent Protein mBanana

mBanana is a novel monomeric red fluorescent protein mutant. It was cloned and expressed in Escherichia coli with 10 histidine residues at its N-terminal. After cleavage of the His tag by TEV protease, the mBanana was further purified and crystallized by the hanging-drop vapor-diffusion technique. The crystals can diffract to 2.0A resolution and one set of completed data was collected. It showed that the orthorhombic mBanana crystal was in space group P21 with unit cell parameters (48.629, 42.667, 61.714, 90, 111.676, 90) and contained one molecule in one asymmetric unit.